The worldwide demand for platelets is increasing, in large part due to their prophylactic use to prevent bleeding in thrombocytopenic cancer patients. The success of new therapies, especially for patients with blood cell cancers, is helping to drive up the number of annual cases of therapy-related thrombocytopenia. Typically patients receive platelet transfusions when their platelet count falls below a “trigger” threshold level and thus the frequency of transfusions in thrombocytopenic patients, in part, depends on the circulating lifespan of the transfused platelets. Interestingly, it has been recently reported that the lifespan of circulating platelets largely depends on the amount of exposed penultimate residues contained in platelet surface glycan structures. On nascent platelets, these residues are normally capped and “masked” by sialic acid. The terminal sialic acid residues on glycan chains can be removed by sialidase enzymes present in blood. Loss of sialic acid on the platelet surface glycans, in turn, then leads to a more rapid clearance of platelets by hepatocytes and macrophages. See Sørenson et al., Blood, 114: 1645-1654 (2009). Accordingly, an agent that could prevent this more rapid in vivo platelet clearance and sustain circulating platelet levels for longer periods of time, should provide a more efficient prophylactic therapy for thrombocytopenic patients.
Storage of platelets for transfusions has long been a difficult issue. According to the Food and Drug Administration's Blood Products Advisory Committee statement, issued Mar. 15, 2002, entitled “Review of Data Supporting Extension of Dating Period for Platelets”: “Bacterial contamination of platelet products continues to be a problem with a contamination rate estimated at 1/2000 units. Storage of platelets at room temperature for up to 5 days allows for proliferation of bacteria in platelet units, and “older” platelets have been associated with increased incidence of septic transfusion reactions. Various approaches are being developed that would either screen or chemically decontaminate platelet units prior to transfusion. If such methods are shown to decrease bacterial contamination of platelet products, storage of platelets out to 7 days may become practical.”
Attempts have been made to reduce the incidence of contamination and extend the storage life of platelets by refrigeration or cold storage. See, Snyder and Rinder, N. E. J. Med. 348:2032-2033 (2003). However, platelets, unlike other transplantable tissues or cell types, do not tolerate refrigeration and disappear rapidly from the circulation if subjected to chilling before transplantation or transfusion. See Rumjantseva et al., Nature Medicine, 15:1273-80 (2009).
Andrews and Berndt, Current Biology, 13:R282-84 (2003) suggest that during chilling of platelets GPIbα could be modified in such a way that cold platelet storage may be feasible by maintaining hemostatic activity and preventing accelerated clearance. However, attempts to inhibit the rapid clearance of long-term stored, chilled platelets have thus far achieved very limited success. For example, Wandall et al., Blood, 111:324956 (2008) report the inability to prevent rapid clearance through galactosylation. Hoffmeister et al., US Patent Application 2008/0138791, report some success in reducing clearance and thereby prolonging the survival of platelets through glycan modification of GPIbα molecules.
The useful life of platelets stored at room temperature remains limited because of the risk of contamination and loss of function. The current inability to chill platelets for longer-term storage, thereby allowing the “stockpiling” of platelets with preserved function, results in chronic shortages of platelets for clinical transfusions and adds to the overall costs of clinical platelet transfusions.
GPIb-IX-V is a multifunctional hetero-complex of four distinct glycoprotein chains, abundantly found on the surface of platelets. The GPIbα chain is one of the subunits of GPIb-IX-V and its N-terminal domain is capable of interacting with several proteins that are either circulating in or exposed to the bloodstream. These proteins include von Willebrand Factor (VWF), thrombin, Factor XI, Factor XII, kininogen, thrombospondin 1 (TSP-1), integrin Mac-1 (CD11b/CD18, αMβ2 or CR3), P-selectin, as well as Ashwell-Morell receptors. Because of this range of interactions, GPIbα has a broad role in platelet function with regard to thrombosis, hemostasis and inflammation. Specific binding events mediated by GPIbα can be separated and vary in importance to hemostatic function. For example the importance of regulated binding to VWF is demonstrated by the finding that a single amino acid substitution in the N-terminal domain of GPIbα can cause gain-of-function phenotypes resulting in human platelet-type von Willebrand disease.
Recent experimental evidence suggests that when platelets are collected and cooled by refrigeration during storage prior to transfusion, the GPIbα glycoprotein chain plays a key role in mediating the subsequent rapid clearance of those transfused platelets from the circulation of the recipient. This rapid clearance has been reported to involve surface clustering of the glycans and protein components of GPIbα on stored platelets, which are observed to form interactions with the recipient's Mac-1 and the Ashwell-Morell asialoglycoprotein receptors. See Rumjantseva et al., Nature Medicine, 15:1273-80 (2009).
Given the role that GPIbα plays with regard to multiple platelet functions, it has been previously contemplated that a specific antagonist to one or more of the GPIbα interaction domains might have therapeutic value in treating cases of undesired thrombosis, inflammation, thrombocytopenia, and rapid platelet clearance. However, experimental attempts using proteins, including antibodies and antibody derived fragments, to block the GPIbα interaction domains have typically resulted in undesired thrombocytopenia. Thus, there exists the need in the art for a therapeutic agent or drug that will serve as a specific GPIbα binding domain antagonist, without causing undesired thrombocytopenia. Moreover, a drug that selectively inhibits certain GPIbα binding functions, yet preserves the capacity of the platelet to maintain its other hemostatic functions, would have substantial therapeutic utility in a variety of vascular disease settings.
The discovery, using a phage display screening approach, of a ten amino acid cyclic peptide termed OS-1, capable of binding to the N-terminal domain of GPIbα was recently reported by Benard et al., Biochemistry, 47:4674-82 (2008). The ability of OS-1 to block VWF-mediated platelet aggregation in vitro, was reported with this peptide. Two other peptides, designated as PS-4 and OS-2, were also shown to competitively inhibit the interaction between VWF-A1-domain and GPIbα. However, no in vivo data was provided in this study and inhibition of Mac-1 binding to GPIbα was not demonstrated. Indeed, simultaneous inhibition of both VWF and Mac-1 binding is unexpected for small peptides. In fact, using small peptides as inhibitors, it has been reported that the binding sites for VWF and Mac-1 on GPIbα are inhibited independently and therefore distinct binding sites. Munday et al., Blood (ASH Annual Meeting Abstracts) 114:472 (2009) and oral presentation Dec. 7, 2009. The N-linked glycans present on GPIbα having exposed βGlcNAc and/or galactose residues represent an entirely separate point of interaction between either GPIbα and Mac-1 or GPIbα and the asialoglycoprotein (Ashwell-Morell) receptors. Given its small size, it is unlikely the OS-1 peptide is able to create a steric interference of this interaction.
McEwan et al., Blood 114:4883-85 (2009) describes the non-covalent interaction of OS-1 peptide with GPIbα and demonstrates that the GPIbα-OS-1 complex structure overlaps with the structure of the GPIbα-VWF A1 domain complex. This indicates that the OS-1 peptide directly interferes with binding of VWF to GPIbα. In commenting on the findings of McEwan, Lopez and Munday, Blood 114:4757-58 (2009) hypothesized that the OS-1 peptide occupies the site where VWF would interact with GPIbα and effects a conformational change in GPIbα that prevents formation of the GPIbα-VWF complex.
There remains a need for improved methods and materials useful for extending the useful storage life of platelets. Such methods should reduce the contamination of platelets, for example, by reducing bacterial and viral growth, yet substantially preserve the platelet's hemostatic function and in vivo half-life after transfusion. There is a further need for methods and materials for the treatment of thrombosis, vascular inflammation, thrombocytopenia, and other platelet-related disorders.